Recovery of enzymes

ABSTRACT

BACTERIAL AMYLASE BACTERIAL PROTESES AND MIXTURES THEREOF ARE RECOVERED FROM A BACTERIAL WHOLE CULTURE FERMENTATION MEDIUM BY A PROCESS WHICH INVOLVES THE ADDITION OF AN INORGANIC SULFITE, PREFERABLY SODIUM SULFITE, WHICH ENHANCE THE YIELD OF ENZYME AS MASURED BY THE ENZYME ACTIVITY, PRODUCES A WHITER PRODUCT, AND INHIBITS SPOLIAGE OF A CONCENTRATED AQUEOUS SOLUTION OF THE ENZYME.

United States Patent U.S. Cl. 195-68 8 Claims ABSTRACT OF THE DISCLOSURE Bacterial amylase, bacterial protease and mixtures thereof are recovered from a bactrial whole culture fermentation medium by a process which involves the addition of an inorganic sulfite, preferably sodium sulfite, which enhances the yield of enzyme as measured by the enzyme activity, produces a whiter product, and inhibits spoilage of a concentrated aqueous solution of the enzyme.

This application is a division of copending application Ser. No. 849,148 filed Aug. 11, 1969, now matured into US. Pat. 3,700,561.

BACKGROUND It is well known that various types of enzymes, which are water soluble, can be produced by fermentation using an aqueous medium containing a microorganism and various nutrients. The microorganisms are available in various culture collections and it is known that certain microorganisms will produce certain enzymes under certain fermentation conditions. The present invention is not concerned with the type of microorganism used or the fermentation conditions.

Once the fermentation has been completed to the desired extent, the whole fermentation culture medium usually consists of a discolored, liquid suspension of finely divided solids which remain suspended and do not settle readily. These suspended solids render the culture medium opaque and are difficult to remove by conventional means such as filtration. The desired enzyme or enzymes, being water soluble, normally remain dissolved in the whole culture. However, they can usually be precipitated by adding a water soluble compound such as, for example, a water soluble salt, which salts out the enzymes. Thus, the addition of water soluble compounds to the whole culture could salt out the enzymes which would then exist in an undissolved state along with a number of contaminants.

Enzymes normally are easily degraded and suffer loss of potency due to heat, moisture and contact with other substances. The preparation of enzymes of high potency therefore become a problem.

In recent years the use of protease in domestic and commercial washing compositions and detergents has increased the demand for high potency purified enzymes. It would therefore be desirable to provide a process for producing protease and other enzymes which facilitates the removal of contaminants from the whole culture, protects the enzymes against loss of potency, produces lighter colored filtrates, afiords control of pH, permits concentration of the filtrates before separation of the enzymes and without spoilage, and affords economical recovery of high potency enzymes.

OBJECTS One of the objects of this invention is to provide a new and improved process for producing enzymes.

Another object is to provide a new and improved process for producing protease and/or amylase.

A further object is to provide a new and improved process for producing a protease and other enzymesby a method wherein cells, proteins, carbohydrates and other contaminants are removed from the whole culture a primary stage while keeping the enzymes in a dissolved state, and the enzymes are removed, ina secondary stage.

Another object is to carry, out a process of the type described in which precipitated solids from the primary stage are readily separated by filtration.

Another object is to produce ligher colored filtrates from the primary stage.

An additional object is to provide an improved method of controlling pH in the primary and secondary stages.

A further object is to provide a new and improved method of preserving enzymes against loss of potency during the recovery process.

Another object is to provide a new and improved process for recovering high potency enzymes as solids.

Other objects will appear hereinafter.

THE INVENTION In accordance with the invention, water soluble enzymes are recovered from a whole fermentation culture medium by a process which involves as a primary stage the addition to the whole culture after fermentation is completed of water soluble inorganic compounds which react with one another or with another water soluble inorganic compound already present in the culture medium to precipitate a water insoluble compound or compounds. This in si precipitate carries down with it various contaminants including water insoluble proteins, cells of microorganisms used in the fermentation, carbohydrates, and other ma-- terials which normally remain suspended in the fermentation medium and are diflicult to remove by the usual methods such as filtration. When combined with the in situ preciptate, these contaminants are readily separated. The residual liquid contains the desired enzyme or enzymes which can be recovered. A further feature of the invention involves a secondary stage in which a solid enzyme is precipitated by adding water soluble compounds, at least one of which is preferably an inorganic sulfite, to the residual liquid from the primary stage.

In the primary stage, the in situ precipitate can be formed by double decomposition by adding two or more Water soluble compounds to the whole culture which react in water to form insoluble precipitates, for example,

calcium chloride and disodium hydrogen ortho phosphate to form tricalcium phosphate; aluminum sulfate and sodium carbonate to form aluminum hydroxide; aluminum sulfate and sodium sulfite; calcium chloride, disodium phosphate, sodium sulfite and aluminum sulfate to form a mixed precipitate; calcium chloride, disodium phosphate and sodium carbonate; calcium chloride and sodium sulfite; and calcium chloride, disodium phosphate, sodium carbonate, aluminum sulfate and sodium sulfite.

In the secondary stage, various water soluble compounds can be added to cause the enzyme to precipitate, for example, sodium sulfate, sodium thiosulfate, sodium sulfite, sodium acid sulfite, sodium metabisulfite, sodium hydrosulfite and mixtures thereof. The addition of a water soluble inorganic sulfite has a special beneficial effect in improving the recovery of enzyme. The presence of the sulfite also makes it possible to concentrate the enzyme solution without spoilage by evaporation under subatmospheric pressures. Without the sulfite, a lower yield of enzyme is obtained as measured by the enzyme activity. The sulfite can also be used to control the pH. The addition of the sulfite in the primary and/or secondary stages also produces a whiter enzyme product. Another advantage of using the sulfite is that a concentrated aqueous solution also be added in the primary stage.

' A preferred method of recovery is to centrifuge the precarbonate. The mixtures were then filtered and the filtrates assayed for protease enzyme activity. The CaCl and the NaH PO were added as 25% (w./v.) solutions, with the cipitated enzyme and mix the wet cake with a suflicient quantity of water absorptive solid diluent to give a dry appearing final product which will normally contain 5% to 7% .water. Examples of solid diluents are calcium acetate monohydrate, zinc oxide, sodium sulfate, magnesium sulfate, sodium tripolyphosphate, aluminum sulfate and mixtures thereof.

7 The pH in the primary and secondary stages Will normally be within the range of 5 to 12, depending upon the enzyme. Thus, one protease whole culture might have a 'pH. of 7.0-7.5 and another a pH of 6.7-7.3. An amylase whole culture might have a pH of 7.3-7.6.

The quantity of precipitants added in the primary and secondary stages should be the minimum amounts which will etfectively bring about the desired precipitation. In the primary stage this will usually not exceed-1% by weight of the whole culture. In any case, excess soluble salts over the amounts required to produce a precipitate by double decomposition should be avoided where the excess is sufficient to salt out the enzyme.

In the secondary stage the quantity of salt added per 'unit volume of liquid is important. Below 20% salt the enzyme precipitation is usually incomplete. The amount of salt used as a precipitant is normally just below saturation solubility at the temperature used.

The temperature in the primary and secondary stages should be below those at which substantial decomposition of the enzyme occurs. With protease, amylase or mixtures thereof the temperatures usually do not exceed 33 C.

The following examples, in which the quantities are by weight unless otherwise indicated, illustrate the practice of the invention in producing protease, amylase and mixtures thereof.

EXAMPLE 1 slurry of calcium hydroxide were added slowly to an agitated bacterial protease fermentation whole culture (2000 ,mL; 5300 PV units per ml.;

10.6 10 PV units) while maintaining the whole culture pH at 8.5 by the simultaneous addition of a 10% (v./w.) aqueous solution of phosphoric acid. After all the calcium hydroxide was added, the mixture was adjusted to a final pH of 6.5 by the further addition of the diluted phosphoricacid. The mixture was then filtered on a Biichner funnel with the addition of 5% (w./v.) Filteraid FW-l4; The resultant filtered cake was washed with distilled water (1500 ml.) and the Wash combined with the filtrate. The combined clear solution totalled 3500 m1. and assayed 2000 PV per ml. (7.0x 10 PV units) for an overall protease enzyme recovery of 66%. Sodium sulfate (125 grams) was added to a portion of the combined solution (500 mi; 2000 PV units per ml.; 1.00X 10 PV units) and the mixture stirred for- 2 hours at room temperature. The precipitate which formed was filtered and washed with a 25% (w./v.) aqueous solution of sodium sulfate *(100 ml.). The filtered wet cake weighed 1.29 grams and assayed 343,000 PV units per gram (0.442 10 PV'units) fora recovery over the precipitation step of 44.2%. The over all recovery from the whole culture to the precipitated wet cake was 29.2%.

EXAMPLE 2 taneous addition of aqueous sodium hydroxide or sodium amount of NaH PO calculated on a stoichiometric basis. The NaH PO solutions were adjusted to pH 7 or 9, as indicated.

The filtrates from the CaCl -NaH PO and the Al (SO treatments were clear While that from the MgSO treatment was turbid. The filtrate from the AI (SO treatment was very much lighter in color than the other filtrates, however, the filtration rate using this salt treatment was much slower.

A 25% (w./v.) Na SO solution was then added to. the combined filtrates from the CaCl -NaH PO treatments, and the precipitated enzyme solid recovered as described in Example 1 except that the wet cake wash step was omitted. A wet cake assaying 763,400 PV units per gram was obtained, with a protease enzyme recovery of 64%.

A 25 (W./v.) Na SO solution was added to the filtrate from the Al (SO treatment and the precipitated enzyme solid recovered in a like manner. A wet cake assaying 771,000 PV units per gram was obtained, with a protease enzyme recovery of 75%. The enzyme wet cake obtained in this manner was very much lighter in color than that obtained from the CaCl -NaH PO treatment.

EXAMPLE 3 A 25% (w./v.) CaCl solution and a 25% (w./v.) Na HPO solution were slowly added to an agitated bacterial protease fermentation whole culture (42 gallons, 6300 PV units per ml., l.00 10 PV units) while maintaining the whole culture pH at 6.3-6.7 by the simultaneous addition of a Na CO solution. The amount of CaCl added was 1% (w./v.) based on the whole culture, with the Na HPO added on a calculated stoichiometric basis. The mixture was then filtered using Filteraid FW- 14 and the filtered cake washed with water. The combined filtrate and wash totalled 98.9 gallons and contained 0.96 10 PV units, for a protease enzyme recovery. of 96%.

EXAMPLE 4 Using the same bacterial protease fermentation whole culture described in Example 3, a similar filtration experi ment was carried out using Al (SO A 15% (W./v.) solution of Al (SO was slowly added to the agitated bacterial protease fermentation Whole culture (43 gallons, 6300 PV units per ml., 1.02 10 PV units) while maintaining the whole culture pH at 63-67 by the simultaneous addition of a Na CO solution. The amount of Al (SO added was 1% (w./v.) based on the whole culture. The mixture was then filtered using Filteraid FW-l4 and the filtered cake washed with water. The combined clear filtrate and wash totalled 98.7 gallons and contained 0.945 10 PV units, for a protease enzyme recovery of 92.7%.

A portion of the filtrate obtained above (10,000 ml., 3150 PV units per ml., 31.5 10 PV) was treated with 2500 grams of Na SO as a 25 (W./v.) solution in the manner indicated in Example 1. The recovered precipitated enzyme solid contained 17.8 10 'PV units, for a protease enzyme recovery of 56.5%.

EXAMPLE 5 The Al (SO filtrate obtained in Example 4 was then treated with Na SO Aliquots of the filtrate (500 ml.; 3300 PV units per ml., 1.65 X PV units) were adjusted Percent protease activity Total sulfite remaining salts added; Temp. after 20 percent (w./v.) C.)

hours to the pH levels shown below using sodium acid sulfate 5 96 8 and the Na SO added as a 25% (w./v.) solution to pre- 1 cipitate the protease enzyme. 82:3 Protease enzyme activity 3;: Filtrate adjusted to in precipitated cake, 10 95.2 indicated pH: percent recovery 32:? 5.5 78.2 87.9 6.0 76.7 333 6.5 72.1 1.0 7.0 70.5 7.5 75.3 7.1

EXAMPLE 6 A bacterial protease fermentation whole culture was treated with 2( 4)s 1n the manner descnbed A bacterial protease fermentation whole culture was E p vvlth a protease enzyme ry 9 995% treated with 1% A1 SO in the manner described in 1n the clear light colored filtrate. The salts indicated be- Example 4 with a protease enzyme recovery of 885% low were then added to aliquots of the filtrate. The mixin the clear fi1t t tllfe?) (P Were held at for 2 hoursend the The following salts were added to aliquots of the filpreclpltated p e y e recovered y filtration-The trate at pH 6.5 and 33" c. and the mixtures held at this Protease enzyme Sohds p p e 1I1 the Presence of the temperature for 2 hours before filtering the precipitated sulfite salts were very much hghter 1n color than those protease enzyme. precipitated by the other salts.

Protease activity Protease activity in precipitated in precipitated cake, percent cake, percent Salts added to filtrate, percent (w./v.) recovery Salts added to filtrate, percent (w.lv.) recovery 25% NEZSOJ 68. c 25% NEESOA 81.6 25% Na2s04 5% Natsos 5% Natsso 92.3 25% NazSO 25% Nassot-t- 3% NazSO 2% N32S205.,-. 93. 25% NazSOa 11.4% Nazszos 90.6 25% Nassotz 35% NaOL 25 +0.51% sodium thiosuliate 86. 7 +10% sodium thiosulfate 84. 7 +25% sodium thiosuliate. 80. 6 +50% sodium thiosulfate 83. 7 EXAMPLE 7 +10.0%s0d1umth1osu1fate 87.8 The bacterial protease filtrate obtained in Example 6 after treatment of the fermentation whole culture with Al (SO was used in a test to evaluate the amount of EXAMPLE 10 sulfite salts which should be added with the sodium sul- USlng the procedure described in Example 4, a 15% fate to precipitate the protease enzyme. The following salt Solution of M26003 was slowly added to an combinations were added to 500 ml. ahquots of filtrate p agltated bacterial protease fermentation whole culture (3300 PV un1ts per ml., 1,650,000 PV units) and the (1000 m1 6170 PV o Units per m1l., 6.l7 10 PV un1ts) mixtures held at 33 C. for 2 hours. The mixtures were while I maintaining the whole culture pH at 6.2 by the simulthen filtered on a Buchner funnel using Filterald FW-ZO. The recovered reci itated enz me cakes contained the taneous addmon of a Nazsos Solution The amount of inflated activmlgs P Y Al (SO added was 1% (w./v.) based on the whole 1 culture. The amount of Na SO used was 2.2% (w./v.) based on the whole culture. The mixture was then filtered on a Biichner funnel using Filteraid FW-20 and the filtered Protease activity in cake washed with water. The combined clear filtrate and Salts added, percent (w./v.) precipitates wash totalled 1230 ml. and contained 608x10 PV units Percent for a protease enzyme recovery of 98.5%. A salt. mixture Naesot NazSO: Nazsgos Total PV recovery consisting of Na SO (50 grams), Na- SO (4.8 grams) 25 o 0 M 1 164 500 m6 and NaHSO (2.8 grams) was then added to an aliquot 25 4 4 0.7 114321250 sea of the filtrate (200 mL; 4700 PV units per ml., 940,000

i 2 i3 i'iii tfig 2%: PV units) and the mixture stirred for 2 hours at 33 C.

25 0 6 0.4 11211250 73.4 The precipitated protease enzyme solid was recovered by 25 0 2 M32150) filtration and contained 860,000 PV units, for a protease enzyme recovery of 91.5% The recovered solid had a very light color.

EXAMPLE 8 EXAMPLE 11 The bacterial protease filtrate obtained in Example 6 Using the Procedure described in Example a 15% after treatment of the fermentation whole culture with sohlheh 1f 2( 4)a wasfilowly added to an A1,,(SO was us d in a li id heat t bilit t t t d agitated bacterial protease fermentation whole culture (90 termine the efiectiveness of the sulfite salts in preventl 587213711111ts P X 109 PV i While ing the loss of protease enzyme activity at elevated temmalhtalhihg the Whole culture P at y the slmllltalleperatures. Filtrate-sulfite salt solutions were prepared as ous addition of a Na SO solution. The amount of shown below and the solutions (pH 6.5) held at the in- Al (SO added was 1% (w./v.) based on the whole dicated temperatures for 20 hours. The sulfite salt mixture culture. The amount of Na SO used was 2.5% (w./v.) consisted of 60% Na SO and 40% Na S O based on the whole culture. The volume of the Whole cul- 7 ture after the addition of the salts was 110 gallons and it assayed 5230 PV units per ml. (2170x10 PV units). The treated whole culture was then filtered on a plate and frame press using Filteraid FW-l4 and washed with water. The combined clear filtrate and wash totalled 192.4 gallons and contained 2.l62 10 PV units. The combined filtrate and wash was then evaporated in vacuo to 78.9 gallons. This concentrate contained 2.050 10 PV units. A salt mixture consisting of NaSO (2500 grams), NaSO (150 grams) and NaHSO (100 grams) was added to an aliquot of the concentrate (10,000 ml.; 6288 PV units/ml., 62.88 10 PV units) and the mixture stirred for 2 hours at 33 C. The precipitated protease enzyme solid was recovered using a Sharples Super Centrifuge. A total of 143 grams of wet solid (42.5% solids) assaying 410,000 PV units per grams (5 8.6x 10 PV units) were obtained, for a protease enzyme recovery of 93.2%. A portion of this wet solid was dried in vacuo at room temperature and a dry protease solid assaying 891,250 PV units per gram (96.2% solids) was obtained. Since the calculated theoretical dry solid potency was 964,700 PV units per gram, the recovery over the drying step was 96.0%.

A 50-gram portion of the centifuged enzyme solid (20.5 10 PV units) was Washed by slurrying the solid in a 25% (w./v.) Na SO solution (1000 ml.) and then recentrifuging the mixture. A total of 37 grams of washed protease enzyme solid (42.0% solids) assaying 600,000 PV units per gram (22.2 x10 PV units) were obtained, for a 108.3% recovery. A portion of this wet solid was dried in vacuo at room temperature and a dry protease solid assaying 1,262,500 PV units/gram (96.4% solids) was obtained. Since the calculated theoretical dry solid potency was 1,428,600 PV per gram, the recovery over the drying step was 91.7%.

EXAMPLE 12 Using the procedure described in Example 4, a 25 (w./v.) CaCl solution (20 ml.) and a 25% (w./v.) Na HPO solution (17.1 ml.) were added to an agitated bacterial protease fermentation whole culture (500 ml.; 7760 PV units per ml., 388x10 PV units) while maintaining the whole culture pH at 6.4 by the simultaneous addition of a 25 Na SO solution (15 ml.). The amount of CaCl added was 1% (W./v.) and the amount of Na HPO added was 0.86% (w./v.), based on the whole culture. A 15% Al (SO 3 solution (50ml.) was then added to the agitated Whole culture while maintaining the whole culture pH at 6.4 by the simultaneous addition of a 25 Na SO solution (80 ml.). The amount of Al (SO added was 1.5% (w./v.) based on the whole culture. The total Na SO added was 4.75% (w./v.) based on the whole culture. The mixture was then filtered on a Buchner funnel. The resultant clear filtrate (690 ml.) asssayed 5920 PV units per ml. (4.08 l PV units), for a protease enzyme recovery of 105.2%.

EXAMPLE 13 Using the procedure described in Example 4, a 25 (w./v.) CaCl solution (20 ml.) and a 15% (w./v.) Al (SO solution (50 ml.) were added slowly to an agitated bacterial protease fermentation whole culture (500 ml.; 7760 PV units per ml., 3.88 X 10 PV units) while maintaining the whole culture pH at 6.4 by the simultaneous addition of a 25 (w./v.) Na SO solution (100 ml.). The amount of CaCl added was 1% (w./v.), the amount of Al (SO added was 1.5% (w./v.) and the amount of Na SO added was (w./V. based on the whole culture. The mixture was filtered on a Biichner funnel. The resultant clear filtrate (680 ml.) assayed 5 632 PV units per ml. (3.83 x PV units), for a protease enzyme recovery of 98.7%.

EXAMPLE 14 Using the procedure described in Example 4, a 15 (w./v.) Al (SO.,) solution (66.6 ml.) was added slowly to an agitated bacterial amylase fermentation whole culture (1000 ml.; 2592' DV units per ml., 2.592 10 DV units; 1325 PV units per ml., 1.325X 10 PV units) while maintaining the whole culture pH at 6.8 by the simultaneous adidtion of 30% (w./v.) Na SO (70 ml.). The amount of Al (SO added was 1% (w./v.) and the amount of Na SO added was 2.1% (w./v.), based on the whole culture. The mixture was then filtered on a Biichner funnel. The resultant clear filtrate (1140 ml.) assayed 1287 DV units per ml. (1.465 X 10 DV units), for an amylase activity recovery of 56.4%.

7 EXAMPLE 15 Using the procedure described in Example 4, a 25 (w./v.) CaCl solution (20 ml.) and a 25% (w./v.) Na H PO solution (17.1 ml.) were added to an agitated bacterial amylase fermentation whole culture(500 ml.; 2592 DV units permL, 1,296 10 DV units; 1325 PV units per ml., 0.663 l0 PV units) While maintaining the whole culture pH at 7.0 by the simultaneous addition of a 25% (w./v.) Na CO solution (20 ml.). The amount of CaCl added was 1% (w./v.), the amount of Na HPO added was 0.86% (w./v.) and the amount of N21 CO added was 1% (w./v.), based on the wholeculture. The mixture was then filtered on a Buchner funnel. The resultant clearfiltrate (560 ml.) assayed 2384 DV units per ml. (1.335 10 DV units) for a amylase activity recovery of 103.0%, and 1225 PV units per ml. (0.686 10 PV units) for a protease activity recovery of 103.5%. A salt mixture consisting of Na SO (37.5 grams) Na SO (6.5 grams) and NaHSO (1.5 grams) was then added to an aliquot of the filtrate ml.; 357,600 DV units; 183,750 PV units) and the resultant mixture stirred for 2 hours at 33 C. The precipitated enzyme solid (1.47 grams) was recovered byfiltration. The wet cake assayed 189,000 DV units per gram (277,830 DV units) for an amylase activity recovery of 77.7%, and 107,000 PV units per gram (157,290 PV units) for a protease activity recovery of 85.6%

EXAMPLE 16 Using the procedure described in Example 4, a 25% (w./v.) CaCl solution (20 ml.) was added to an agitated bacterial amylase fermentation whole culture (500 ml.; 2592 DV units per ml., 1.296 10 DV units; 1325'PV units per ml.; 0.663 (10 PV units) while maintaining the whole culture pH at 7.0 by the simultaneous addition of a 25 (w./v.) Na SO solution (22.7 ml.). The amount of CaCl added was 1% (w./v.) and the amount of Na SO was 1.13% (w./v.) based on the whole culture. The mixture was thenfiltered on a Buchner funnel." The resultant clear filtrate (550 ml.) assayed 2353 DV units per ml. (1.294 10 DV units) for an amylase activity recovery. of 99.8%, .and 1 PV units per m1. (0.649 10 PV units) for a protease activity recovery of 97.9%. A salt mixture consisting of Na SO (50 grams), Na SO (6 grams) and NaHSO (2 grams) was then added to an aliquot of the filtrate (200 ml.; 470,600 DV units; 236,000 PV units) and the mixture stirred for 2 hours at 33 C. The precipitated solid was recovered by filtration (1.95 grams) and assayed 200,000 DV units per gram (390,000 DV) for an amylase activity recovery of 82.9%, and94,'000 PV'unitsper gram (183,300 PV) for a protease activity recovery of 77.7%

EXAMPLE 17 Using the procedure described inExample 4, a 25% (w./v.) CaCl; solution (20 ml.) and a 25% (w./v.) Na2HP04 solution 17.1 ml.) were added to an agitated bacterial amylase fermentation whole culture (500 ml.;

2592' DV units per ml., 1.296X105DV units; 1325 -PV added was 0.75% (w./v.), based on the whole culture. The mixture was filtered on a Buchner tunnel. The resultant clear filtrate (560 ml.) assayed 2353 DV units per ml. (1.318 DV units) for an amylase activity recovery of 101.7%, and 1375 PV units per ml. (0.770 10 PV units) for a protease activity recovery of 116.1%. A salt mixture consisting of Na SO (50 grams), Na SO (6 grams) and NaHSO (2 grams) was then added to an aliquot of the filtrate (200 ml., 470,600 DV units; 275,000 PV units) and the mixture stirred for 2 hours at 33 C. The precipitated solid was recovered by filtration (1.70 grams wet cake) and assayed 239,000 DV units per gram (406,300 DV) for an amylase activity recovery of 86.3%, and 147,000 PV units per gram (249,900 PV) for a protease activity recovery of 90.9%.

EXAMPLE 18 Using the procedure described in Example 4, a 25% (w./v.) CaCl solution ml.) and a (w./v.) Na HPO solution (17.1 ml.) were added slowly to an agitated bacterial amylase fermentation whole culture (500 ml.; 2592 DV units per ml., l.296 10 DV units; 1325 PV units per ml., 0.663 10 PV units) while main taining the whole culture pH at 7.0 by the simultaneous addition of a 25% (w./v.) Na SO solution (60 ml.). Then a 15% (w./v.) AI (SO solution (33.3 ml.) was added slowly to the agitated whole culture while maintaining the whole culture pH at 7.0 by the simultaneous addition of a 25% (w./v.) Na SO solution (60 ml.). The amount of CaCl, added was 1%, the amount of Na HPO added was 0.86%, the amount of Na CO added was 1%, the amount of Al (SO added was 1% and the amount of Na SO added was 3%, based on the whole culture. The mixture was filtered on a Buchner funnel. The resultant clear filtrate (660 ml.) assayed 1848 DV units per ml. (1.220 10 DV units) for an amylase activity recovery of 94.1%, and 1180 PV units per ml. (0.779 10 PV units) for a protease activity recovery of 117.5%. A salt mixture consisting of Na SO (62.5 grams), Na SO (3.0 grams) and NaHSO (2.0 grams) was added to an aliquot of the filtrate (250 ml.; 462,000 DV units; 295,000 PV units and the mixture stirred for '2 hours at 33 C. The precipitated solid was recovered by filtration (1.65 grams wet cake) and assayed 255,000 DV units per gram (420,750 DV units) for an amylase activity recovery of 91.1% and 155,000 PV units per gram (255,750 PV units) for a protease activity recoveryof 86.7%.

EXAMPLE 19 Using the procedure described in Example 11, a bacterial protease fermentation whole culture was processed to yield a centrifuged protease enzyme wet solid assaying 490,000 PV units per gram. Portions of the wet solid were then mixed with various salts to give dry solid blends, eliminating the need to go through a drying step.

Protease activity, PV/g.

Theory Found 30, see

6 Calcium acetate 40, 666 2 5 Sodium thimnlfnfn 15 Sodium tripolyplhosphate 2 Protease wet so d 6 Calcium acetate liate 15 Sodium tripolyphosphate The invention makes it possible to economically process and produce high potency enzymes such as, for example, bacterial protease, bacterial amylase, and mixtures of bacterial protease and bacterial amylase, from whole cultures, usually obtained by fermentation with Bacillus subtilis in a nutrient medium and containing water, together with the enzymes dissolved in the wa ter, and cells which remain from the microorganisms as well as soluble or insoluble proteins, carbohydrates, and small amounts of salts. These whole cultures are normally brown, turbid and opaque. It is essential from a practical standpoint to recover the enzymes in as light a state as possible. The reaction of two or more compounds in the primary stage, for example, calcium chloride and monosodium or disodium phosphate and/or aluminum sulfate and sodium carbonate to form water insoluble precipitates causes coprecipitation of the cells and other byproducts. By adding sodium sulfite or other water soluble inorganic sulfite in the primary stage the pH can be raised to a range where the enzymes are least subject to degradation, usually at least 6.5 and preferably 6.5-7.6, while the sulfite itself affords enhanced protection against degradation. The precipitated solids can then be separated by filtration or otherwise and the residual liquid processed in the secondary stage.

In the secondary stage, it is desirable first to concentrate by evaporation, usually under vacuum and at a temperature insufiiciently high to degrade the enzyme, for example, not in excess of 33 C. The main reason for this is that the filter cake or centrifuge cake from the primary stage is washed with water to remove any entrained or adsorbed enzymes. The washings are added to the filtrate which increases the volume of the residual. This could be further processed as such but would require the addition of larger quantities of salts. Hence, by concentrating it two or three times the process is more economical. However, too much concentration should be avoided because other substances may be occluded when the enzyme is precipitated. The addition of sodium sulfite, sodium bisulfite, sodium hydrosulfite, sodium hyposulfite, sodium sulfate, sodium thiosulfate, zinc chloride, zinc sulfate, and/or zinc oxide serves several purposes as previously indicated. The sulfites can be used to control the pH, usually within the range of 6.5-7.6. They also protect the enzymes against degradation. All of the soluble salts have a salting out effect. The zinc compounds modify the physical form of the enzyme particles and make them easier to separate from the liquid.

I claim:

1. In the recovery of an enzyme from the group consisting of bacterial amylase, bacterial protease, and mixtures thereof from an aqueous liquid, the process which comprises precipitating said enzyme in the presence of an inorganic sulfite while maintaining a pH within the range of 5 to 12.

2. In the recovery of an enzyme from the group consisting of bacterial amylase, bacterial protease and mixtures thereof from an aqueous whole culture fermentation medium, the process which comprises precipitating the enzyme in the presence of sodium sulfite while maintaining a pH within the range of 5 to 12.

3. A process as claimed in claim 1 in which said enzyme comprises protease.

4. A process as claimed in claim 1 in which said enzyme comprises amylase.

5. A process as claimed in claim 1 in which an inorganic zinc compound is added to said aqueous liquid.

6. A process as claimed in claim 1 in which an inorganic soluble zinc salt is added to said aqueous liquid.

7. A process as claimed in claim 1 in which zinc oxide is added to said aqueous liquid.

1 1 8. A process as claimed in claim 1 in which said aque- 3,141,832 7/1964 ous liquid containing said enzyme is concentrated in the 3,031,380 4/1962 presence of said sulfite. 3,284,316 11/ 1966 3,524,798 8/1970 References Cited 5 UNITED STATES PATENTS 3,700,561 10/1972 Zifier 195-66 R 1 3,627,640 12/1971 Blumberg et a1 19568 195- 66R 12 Burdick 195- 63 X Minagawa et a1. 195--66 R Cayle 195-68 X Lloyd et a1 195-68 X DAVID M. NAFF, Primary Examiner us. 01. X.R. 

